Making Swaps Until Activity Drops - Localizing the Different Specific Activity of Presenilin Homologues to Protein Domains

Abstract

Alzheimer‘s Disease (AD) is the leading cause of dementia and remains a challenge for the scientific community worldwide. γ-Secretase, a membrane spanning aspartic protease complex, is thought to play a crucial role in AD pathogenesis. By the cleavage of the β-amyloid precursor protein (βAPP), γ-secretase generates Aβ fragments, predominantly Aβ40 and Aβ42. The longer Aβ fragments such as Aβ42 are more prone to aggregate to amyloid plaques, which are found in the brains of AD patients. Presenilin (PS) is the catalytic subunit of the γ-secretase complex with two mammalian homologues (PS1 and PS2) present in the cells. While these complexes generate similar ratios of Aβ42 to Aβ40, PS1 containing γ-secretase complexes were shown to have a higher specific activity for the cleavage of βAPP. However, the regions responsible for the difference in activity between the two multi-membrane spanning homologues have not been identified so far. By systematically swapping single or multiple transmembrane domains from PS1 to PS2 and vice versa, this study aims to localize important domains that are responsible for the higher specific activity of PS1. To generate the chimeric PS constructs, the respective cDNAs were cloned and stably transfected into HEK293 cells stably expressing the Swedish mutant βAPP (swAPP). swAPP shows 6-8-fold increase in Aβ production compared to cells expressing normal βAPP, enabling us to analyze effects arising from our chimeric constructs on Aβ generation directly from conditioned media via immunoblotting and ELISA. Additionally, to analyze intracellular cleavage products, a well-established in vitro γ-secretase assay was performed. We expect that localized, non-conserved domains are responsible for the different activity of the two presenilin homologues. Mapping these domains will help us to shed further light on the development and pathogenesis of AD.